Project Summary According to the World Health Organization approximately 35 million people worldwide were living with HIV-1 at the end of 2014 and 1.2 million people died from this disease during the same year. Vaccination is the most effective strategy to prevent infectious diseases but over 25 years of research has failed to generate one against HIV-1. The correlate of protection for most existing vaccines is the development of neutralizing antibodies (1). Due to the genetic variability of HIV-1, a successful vaccine will however need to elicit broadly neutralizing antibodies (bnAbs) that target conserved epitopes on the envelope glycoprotein (Env) (4). Several of these antibodies have been isolated from HIV-1 infected individuals and, upon passive transfer into non- human primates or genetically engineered humanized mice, these antibodies protect against an infection with chimeric simian/human immunodeficiency virus (SHIV) or HIV-1, respectively (5-9). Using immunoglobulin knock-in mice expressing the predicted germline version of a selected bnAb, the proposed research aims to determine the antigenic requirements for a vaccine that can elicit bnAbs in humans. During the K99 phase, I will use these mice to develop a vaccination strategy that can drive the maturation of a specific bnAb from the activation of its predicted germline expressing nave B cell. The knock-mice will be immunized with engineered Env-based antigens developed in collaboration with Dr. Rogier Sanders at the University of Amsterdam. Antigen-specific B cell responses will be analyzed in detail by flow cytometry and single B cell sorting, as well as cloning and sequencing of individual B cell receptors, as previously established in our laboratory (12). Serum and monoclonal antibodies will be evaluated for neutralization against HIV-1 in collaboration with Dr. Michael Seaman at the Beth Israel Deaconess medical Center. The K99 phase will be conducted in the Laboratory of Dr. Michel Nussenzweig at The Rockefeller University. Dr. Nussenzweig has long-standing experience in the field of B cell research. More recently, Dr. Nussenzweig has applied his knowledge to study B cell responses against HIV-1. He has mentored a number of successful postdocs, who now have their own careers as independent investigators. The Rockefeller University provides an outstanding environment for the proposed research and for young scientists to prepare for a career as independent investigators. Apart from gaining additional laboratory skills, mentorship experience and support in future job-search from Dr. Michel Nussenzweig and his laboratory, I plan to attend courses and workshops organized by the Rockefeller University and/or the tri-institutional environment including Memorial Sloan-Kettering and Weill Cornell Medical College with the aim to become a successful independent investigator. During the R00 phase, promising vaccine regimens tested in immunoglobulin knock-in mice will be tested and adapted to animals that have polyclonal immune systems including humanized mice expressing the full complement of human antibody germline genes. The information obtained from the experiments outlined in this proposal will be critical to develop a protective vaccine against HIV-1.